Thermostable polymerases having altered fidelity and methods of identifying and using same

Abstract

The present invention provides a method for identifying a thermostable polymerase having altered fidelity. The method consists of generating a random population of polymerase mutants by mutating at least one amino acid residue of a thermostable polymerase and screening the population for one or more active polymerase mutants by genetic selection. For example, the invention provides a method for identifying a thermostable polymerase having altered fidelity by mutating at least one amino acid residue in an active site O-helix of a thermostable polymerase. The invention also provides thermostable polymerases and nucleic acids encoding thermostable polymerases having altered fidelity, for example, high fidelity polymerases and low fidelity polymerases. The invention additionally provides a method for identifying one or more mutations in a gene by amplifying the gene with a high fidelity polymerase. The invention further provides a method for accurately copying repetitive nucleotide sequences using a high fidelity polymerase mutant. The invention also provides a method for diagnosing a genetic disease using a high fidelity polymerase mutant. The invention further provides a method for randomly mutagenizing a gene by amplifying the gene using a low fidelity polymerase mutant.

1. A composition comprising a Taq DNA polymerase mutant, wherein said polymerase mutant comprises one or more amino acid substitutions selected from the group consisting of Phe667Leu; Asn666Asp; Asn666Ile; Ile665Leu; Leu670Val; Arg660Tyr; Arg660Ser; Gly668Arg; Arg660Lys; Gly668Ser; Gly668Gln; Thr664Ile and Asn666Asp; Ala661Ser and Val669Leu; Ala661Glu, Ile665Thr, and Phe667Leu; and Thr664Pro, Ile665Val and Asn666Tyr, and a template neucleic acid molecule. 
     
     
       2. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Phe667Leu. 
     
     
       3. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Asn666Asp. 
     
     
       4. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Asn666Ile. 
     
     
       5. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Ile665Leu. 
     
     
       6. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Leu670Val. 
     
     
       7. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Arg660Tyr. 
     
     
       8. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Arg660Ser. 
     
     
       9. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Gly668Arg. 
     
     
       10. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Arg660Lys. 
     
     
       11. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Gly668Ser. 
     
     
       12. The composition of  claim 1 , wherein said polymerase mutant comprises the amino acid substitution Gly668Gln. 
     
     
       13. The polymerase mutant of  claim 1 , wherein said polymerase mutant comprises the amino acid substitutions Thr664Ile and Asn666Asp. 
     
     
       14. The polymerase mutant of  claim 1 , wherein said polymerase mutant comprises the amino acid substitutions Ala661Ser and Val669Leu. 
     
     
       15. The polymerase mutant of  claim 1 , wherein said polymerase mutant comprises the amino acid substitutions Ala661Glu, Ile665Thr, and Phe667Leu. 
     
     
       16. The polymerase mutant of  claim 1 , wherein said polymerase mutant comprises the amino acid substitutions Thr664Pro, Ile665Val and Asn666Tyr.